Relationship between histone H3K36 trimethylation and acute renal injury induced by ischemia/reperfusion

AIM: To observe the expression change of H3K36 trimethylation (H3K36me3) in acute kidney injury (AKI) induced by ischemia/reperfusion (IR) and analyze its correlation with SMYD2 and renal injury, and to provide a new target for clinical treatment of IR-AKI. METHODS: The ICR mice (n=30) were randomly divided into IR group (n=15) and sham operation group (sham group, n=15). The AKI model was established by clamping bilateral renal pedicles for 45 min. The serum and renal tissues of the mice were collected after the model was established for 24 h. The levels of blood urea nitrogen (BUN) and serum creatinine (SCr) were detected by biochemical method. The pathological changes of the kidney were observed by HE staining. The changes of NGAL and cleaved caspase-3 were observed by immunohistochemical (IHC) staining. The protein levels of NGAL, SMYD2, H3K36me3, p-P53, P53, cleaved caspase-3, Bax, Bcl-2, STAT3, p-STAT3, JNK and p-JNK1/2/3 in the renal tissues were determined by Western blot. The relationship between H3K36me3 and SMYD2/NGAL was analyzed by Pearson correlation method. RESULTS: Compared with sham group, BUN and SCr levels of the mice in IR group were significantly increased (P<0.05). HE staining showed significant edema, abscission and necrosis in renal tubular epithelial cells of the micc in IR group, and abscission of brush border and large amount of cell debris in the lumen were also observed. The IHC staining results showed that the protein levels of NGAL and cleaved caspase-3 in IR group were significantly increased. The results of Western blot showed that the protein levels of NGAL, SMYD2, H3K36me3, p-P53, cleaved caspase-3, Bax, STAT3, p-STAT3, JNK and p-JNK1/2/3 in IR group were significantly up-regulated (P<0.05), while the Bcl-2 levels were significantly down-regulated (P<0.05). The correlation analysis showed that H3K36me3 was positively correlated with the levels of SMYD2 and NGAL. CONCLUSION: H3K36me3 is closely related to SMYD2 and renal injury in IR-AKI mice, and the up-regulated expression of H3K36me3 may be involved in the regulation of IR-AKI occurrence and development together with the activation of STAT3 and JNK signaling pathways.     

Analysis of different methylation patterns of histone H3 between androgen-dependent and androgen-independent prostate cancer cells by heavy methyl SILAC

AIM：To study the epigenetic mechanisms involved in the evolution of prostate cancer from an androgen-dependent state to an androgen-independent state, and the global difference of histone H3 methylation between androgen-dependent and -independent prostate cancer cells. METHODS：The methylation sites and patterns of histone H3 in androgen-dependent prostate cancer cell line LNCaP and androgen-independent prostate cancer cell line DU145 were analyzed by heavy methyl stable isotope labeling with amino acids in cell culture (SILAC) coupled with 2D LC-MS/MS. Western blotting was used to verify the results from MS. The differential expression of related methylases and demethylases was tested by real-time PCR. RESULTS：Five methylation sites on histone H3 were found in both cell lines, the patterns of which were as follows: H3K14me2, H3R17me1, H3K36me1, H3K36me2, H3K36me3, H3R72me2, H3K79me1 and H3K79me2. There were 2 different peptides both containing methylated H3K36, “KSAPATGGVKKPHR” and “KSAPSTGGVKKPHR”, which were different from the 31th amino acid residue “A” and “S”. The former peptide belonging to histone H3 variants, H31T, H31 and H32, was mainly identified in DU145 cells, the total peptide counts of which were much more than that of the latter peptide belonging to histone H3 variant H31T, suggesting that these 2 cell lines expressed different histone H3 variants. Mono- and dimethylation of H3K36 were not different between these 2 cell lines, but the trimethylation was significantly higher in DU145 cells than that in LNCaP cells. Many H3K36 demethyltransferases were decreased in DU145 cells compared with LNCaP cells. CONCLUSION：The differential expression of histone H3 variants and H3K36 demethyltransferases may result in up-regulation of H3K36 tri-methylation during the evolution of prostate cancer from an androgen-dependent state to an androgen-independent state.     

Advances in modulation of microRNA and DNA methylation in congenital heart diseases

Aberrations in microRNA (miRNA) expression and DNA methylation are causal factors for congenital heart diseases (CHD), which belongs to epigenetic mechanisms. Complex modulation of miRNA on DNA methylation is a critical regulator of gene expression, leading to disease development. The aim of this review is to provide recent progress in the regulation of miRNA and DNA methylation in CHD.     

Changes of DNA methylation at CpG sites in promoter region of TNF-α gene in DENV2-infected peripheral blood mononuclear cells

AIM：To examine DNA methylation at CpG sites in the promoter region of tumor necrosis factor-alpha (TNF-α) gene in dengue virus type 2 (DENV2)-infected peripheral blood mononuclear cells (PBMC).
METHODS：DNA methylation in the promoter region of TNF-α gene was measured by bisulfite sequencing PCR.
RESULTS：The promoter region of TNF-α gene was from -294 bp to +58 bp, including 11 CpG sites. The PCR products identified by aga-rose gel electrophoresis were consistent with the theoretical size. Two sites were methylated at 0 h and 6 h and 6 sites were methylated at 12 h in TNF-α gene promoter region in DENV2-infected PBMC. The average methylation rates were 103%, 121% and 255% at 0 h, 6 h and 12 h, respectively. Significant differences between 0 h and 12 h and between 6 h and 12 h were observed.
CONCLUSION：The DNA methylation in the promoter region of TNF-α gene is increased in DENV2-infected PBMCs.     

庆祝《园艺学报》创刊50 周年

今年是《园艺学报》创刊发行50周年。50年来,《园艺学报》坚持为学术交流服务,为促进学科发展作贡献的办刊原则,以"科学性;创新性;对生产和科研有参考启迪作用"的标准,收录和发表了大量高水平的论文,记载了几代科技工作者呕心沥血创新之作,反映了中国园艺科学技术和园艺产业的发展历程。     

Changes of urinary exosomal leucine aminopeptidase and dipeptidyl peptidase 4 in diabetic nephropathy

AIM: To investigate the changes of urinary exosomal enzymes and the correlation with diabetic nephropathy.METHODS: Thirty-four healthy volunteers and 127 patients of type 2 diabetes mellitus (T2DM) were included in the study. The healthy volunteers served as control. The patients with T2DM were divided into 3 groups based on their 24 h urinary albumin/creatinine ratio (UACR): 50 patients with microalbuminuria in early DN group (DN1), 34 patients with macroalbuminuria in overt DN group (DN2) and 43 patients without albuminuria in DM group. The levels of urine exosomal leucine aminopeptidase(exosome-LAP) and exosomal dipeptidyl peptidase 4(exosome-DPP4) were determined by enzyme-linked immunosorbent assay (ELISA). The following methods were used to determine the biochemical parameters: liquid chromatography for glycated hemoglobin (HbA1c), chemical modification method for cholesterol (CH), Jaffe-kinetic assay for creatinine (CR) and urease-GLDH method for blood urea nitrogen (BUN). Multiple stepwise linear regressions were used to analyze the relationship of exosome-LAP or exosome-DPP4 with HbA1c, CH, UACR, CR and BUN. RESULTS: The levels of exosome-LAP and exosome-DPP4 in DM, DN1 and DN2 groups were significantly higher than those in control group (P<0.01). The exosome-LAP in DN2 group was significantly higher than that in DM group. Correlation analysis showed that the levels of urinary exosome-LAP and exosome-DPP4 were positively correlated with HbA1c, CH, UACR, CR and BUN. Multiple stepwise linear regression analysis showed that CH and UACR were independent determinants for exosome-LAP (P<0.01), and UACR and HbA1c were independent determinants for exosome-DPP4 (P<0.01). CONCLUSION: Urine exosome-LAP and exosome-DPP4 are correlated with the severity of diabetic nephropathy. These parameters may serve as clinical markers for the diagnosis and prognosis evaluation of diabetic nephropathy.     

Effect of PTP4A3 gene on tumor growth and metastasis

PTP4A3 is an oncogene, which encodes phosphatase of regenerating liver (PRL)-3 protein that is a metastasis-associated phosphorylase. At present, a number of studies have found that it promotes cell proliferation, cell motility, cell invasion, tumor metastasis and epithelial-mesenchymal transition. Therefore, it is inseparable with the occurrence and development of cancer. Here, we summarize the relationship betweenPTP4A3 gene and the occurrence and development of cancer, the alteration ofPTP4A3gene expression and the functional role ofPTP4A3 gene in a variety of cancers. This review will help us to understand the correlation betweenPTP4A3 gene and cancer as well as the mechanism of signaling pathway, providing new insights ofPTP4A3 gene targeting strategy for treating cancer.     

Relationship between genetic instability of nm23H1 gene and clinical pathological characteristic in hepatocellular carcinoma

AIM: The aim of this study was to examine the microstatellite instability (MSI) and loss of heterozygosity(LOH) of locus D17S396 on chromosome 17 and their influence on the expression of nm23H1 in hepatocellular carcinoma (HCC),which may provide experimental evidence for the mechanism of nm23H1 gene and tumor metastasis.METHODS: Techniques such as DNA extraction from formalin-fixed paraffin-embedded tissues,PCR-SSCP,ordinary silver stain were used to study MSI and LOH of locus D17S396.Envision immunohistochemistry and Leica-Qwin computer imaging techniques were used to assess the expression of nm23H1.RESULTS: ① The frequency of heredity instability of HCCs was 35.42%.The frequency of LOH in the cases with lymph node or distant organs metastasis or not and with intrahepatic metastasis or embolus of portal vein or not was significantly different (P<0.01),it was higher in stage TNM Ⅲ than that in stageⅠ and Ⅱ.Moreover,it was higher in high tendency to invasion or metastasis cases than that in the low tendency cases (P<0.01).② The expression of nm23H1 was 56.25%.It was significantly different in Edmondson grade,TNM stage and in lymph node or distant organ metastasis cases (P<0.01).The cases with high tendency of invasion or metastasis exhibited lower nm23H1 expression compared with low tendency cases (P<0.01).③ The positive rate of nm23H1 protein in LOH positive group was lower than that in LOH negative group (P<0.05).CONCLUSION: The results indicate that both MSI and LOH of nm23H1 gene control the development of HCC independently in different pathways.LOH inhibits the expression of nm23H1,which endows it with high aggressive and poor prognosis.Increase in the amount of nm23H1 protein expression effectively restrains the tendency to invasion or metastasis of HCCs and improves prognosis of patients.     

Relationship between genetic instability of nm23H1 gene and clinical pathological behaviors in gastric cancer and colonic cancer

AIM: To study the relationship between genetic instability of nm23H1 gene and clinical pathological behaviors in Chinese with gastric cancer and colonic cancer, and provide experimental basis for the mechanism of nm23H1 gene and tumor metastasis. METHODS: This study was conducted on 40 gastric carcinomas and 30 colonic carcinomas. Techniques such as DNA extraction from formalin-fixed paraffin-embedded tissues, PCR-SSCP, ordinary silver stain were used to study microsatellite instability (MSI) and loss of heterozygosity (LOH) of locus D17S396. Envision immunohistochemistry and Leica-Qwin computer imaging techniques were used to assess the expression of nm23H1 protein. RESULTS: In both gastric cancer and colonic cancer, the frequency of MSI was higher in TNM stageⅠandⅡthan that in stage Ⅲ and Ⅳ, while LOH was just opposite. Moreover, the frequency of LOH in lymph node metastasis cases was significantly higher than that without lymph node metastasis cases. The positive frequency of nm23H1 protein with lymph node metastasis was lower than that without lymph node metastasis cases. TNM stage III and IV also exhibited lower nm23H1 protein positive frequency compared with stage I and II. CONCLUSION: MSI and LOH can control the carcinogenesis and metastasis of gastric cancer and colonic cancer through different approaches. MSI may be an early period molecule marker of gastric cancer and colonic cancer. In contrast, LOH appears mostly in the late period of gastric cancer and colon cancer, indicating a high aggressive and poor prognosis.     

红穗和白穗醋栗F3H的克隆及其在果实成熟过程中的表达分析

以红穗醋栗（Ribes rubrum L.）和白穗醋栗（R. albrum L.）果实为试材,采用RACE方法克隆黄烷酮3–羟化酶（F3H）基因cDNA全长序列,分别命名为RrF3H和RaF3H（KU984435和KU984436）。RrF3H全长1 351 bp,开放阅读框长1 098 bp,编码365个氨基酸;RaF3H全长1 291 bp,开放阅读框长1 098 bp,编码365个氨基酸。氨基酸多序列比对表明该基因编码的蛋白具有非血红素双加氧酶结构域（DIOX-N superfamily）和典型的F3H蛋白功能结构域（2OG-FeⅡ_Oxy加氧酶结构域）,属于双加氧酶超家族。系统发育分析表明,RrF3H和RaF3H在进化上具有明显种属特性,属于相对独立的进化分支。定量PCR分析表明,F3H在红穗醋栗中表达量远远高于白穗醋栗,在红穗醋栗中随着果实着色加深表达量逐渐上升,果实着色约75%时表达量最高,之后下降;白穗醋栗中随着果实生长,该基因的表达下降,花色苷含量也呈下降趋势,说明F3H基因在醋栗果实着色过程中发挥作用。     

Correlation between progression of atherosclerosis accelerated by emotional stimulation and imbalance of SDF-1/CXCR4 in mice

AIM To observe effects of emotional stimulation on expression of stromal cell-derived factor-1（SDF-1） and CXC chemokine receptor 4 （CXCR4） in plasma, platelets, aortas, hippocampus and bone marrow of apolipoprotein E gene knockout （ApoE^-/-） mice, and to reveal the possible mechanism of the aggravated atherosclerotic plaque vulnerability by emotional stimulation. METHODS Thirty 8-week-old male ApoE^-/- mice were randomly divided into normal control group, high fat group, and emotional stimulation group. Ten 8-week-old inbred C57BL/6J mice served as blank control group. After 12 weeks of intervention, the serum levels of SDF-1 and CXCR4 were investigated by ELISA. The protein levels of SDF-1 and CXCR4 in platelets, aortas, hippocampus and bone marrow were determined by Western blot. The pathological damage of aortas was observed by oil red O staining. RESULTS Compared with blank control group, normal control group and high fat group, the mice subjected to emotional stimulation showed more serious atherosclerosis in aortas detected by oil red O staining, and increased levels of SDF-1 and CXCR4 in the plasma and aortas were also observed （P<0.05）. The results of Western blot showed that the protein levels of SDF-1 and CXCR4 in platelets, aortas and hippocampus were increased in the mice subjected emotional stimulation, but the expression of SDF-1 and CXCR4 in the bone marrow was decreased （P<0.05）. CONCLUSION Imbalance of SDF-1/CXCR4 may be the key target by which emotional stimulation accelerates the progression of atherosclerosis.     

Detection of EML4-ALK fusion gene and analysis of its clinical features in NSCLC patients with EGFR mutation

AIM: To detect the frequency of EML4-ALK fusion gene in non-small-cell lung cancer (NSCLC) patients who had epidermal growth factor receptor (EGFR) mutation, and to analyze the relationship between EML4-ALK fusion gene and clinical features. METHODS: One hundred and two Chinese patients with NSCLC were selected on the basis of one or more of the following characteristics: female, never/light smoking history and adenocarcinoma histology. The EML4-ALK fusion gene was identified by single-tube multiplex RT-PCR. In EML4-ALK-positive samples, exon 18 to 21 EGFR mutations and exon 1 and 2 KRAS (Kirsten rat sarcoma) mutations were detected by DNA direct sequencing after PCR amplification. RESULTS: Eight specimens (7.8%) were identified with EML4-ALK fusion genes from 102 NSCLC tissues. Of all positive cases, 7 were variant 1 (V1) and 1 was variant 2 (V2). In 8 EML4-ALK-positive samples, exon 18 to 21 EGFR and exon 1 and 2 KRAS were wild-types. Among 8 EML4-ALK-positive cases, 5 cases (5/8, 62.5%) were younger than the mean age. Six cases (6/8, 75%) were female and 7 cases (7/8, 87.5%) were non-smokers. Five cases (5/8, 62.5%) had adenocarcinoma histology. CONCLUSION: EML4-ALK fusion gene defines a new molecular subset of NSCLC with distinct characteristics. The EML4-ALK fusion gene is mutually exclusive to EGFR and KRAS mutations.     

Methylation of p15INK4B gene and its mechanism in patients with myelodysplastic syndrome and leukemia

AIM: To explore the relationship between hypermethylation of p15INK4B gene and the pathogenesis of hematopoietic malignances. METHODS: The expression and methylation of p15INK4B gene and the expression of DNA methyltransferase genes (DNMTs) in bone marrow cells from 54 cases with hematopoietic malignances were detected by RT-PCR, Western blot, and methylation-specific PCR. RESULTS: The p15INK4B gene was methylated more often in high-risk myelodysplastic syndrome (MDS) patients, patients at blast phase of chronic myeloid leukemia (CML-BP) and acute leukemia patients than that in low-risk MDS patients (P<0.01). The expression levels of DNMT_3A and DNMT_3B in acute leukemia patients, high-risk MDS patients, and CML-BP patients were higher than that in low-risk MDS patients (P<0.05). CONCLUSION: The hypermethylation of p15INK4B gene may be one of the most common genetic event in pathogenesis of acute leukemia, high-risk MDS, and blast phase of chronic myeloid leukemia. Furthermore, DNMT_3A and DNMT_3B are substantially over-expressed in the bone marrow cells of these patients.     

香蕉品种对枯萎病菌感病性与对病菌毒素敏感性之间的相关性

为明确香蕉品种对香蕉枯萎病菌感病性与对病菌粗毒素敏感性之间的相关性,采用根部病菌孢子接种测定了36份香蕉品种苗对香蕉枯萎病菌4号生理小种的感病性,以及采用植株根部和叶片毒素处理测定了5份香蕉品种苗对病菌粗毒素的敏感性。结果表明,天宝角蕉(AAA)、柴蕉(AAA)、广粉1号(AAB)和贡蕉(AA)表现抗病,哥斯达黎加蕉(AAA)和抗枯5号(AAA)表现中抗;感病品种对毒素的敏感性高于抗病品种。通过香蕉不同品种的病情指数、萎蔫指数和叶片损伤面积的差别分析,发现香蕉苗期对枯萎病菌感病性与对病菌粗毒素敏感性之间存在正相关性,60d龄期的假植苗病情指数与其叶片损伤面积之间存在显著的正相关性。因而叶片毒素处理法测量香蕉假植苗中部叶片损伤面积可作为一种快速区别香蕉抗病能力的有效方法。     

Involvement of TLR4/NLRP3 inflammasome in contrast medium-induced inflammation and injury in renal tubular epithelial cells

AIM: To investigate whether Toll-like receptor 4 (TLR4) and Nod-like receptor protein 3 (NLRP3) inflammasome were involved in contrast medium (CM)-induced inflammation and injury in renal tubular epithelial cells. METHODS: Iopromide was used to injure NRK-52E cells in the study. The cell viability was measured by CCK-8 assay. The protein levels of TLR4, NLRP3, apoptosis-associated speckle-like protein (ASC), caspase-1 and cleaved caspase-3 were determined by Western blot. The releases of interleukin (IL)-1β and IL-18 were detected by ELISA. The apoptotic rate was evaluated by Hoechst staining, and mitochondrial membrane potential (MMP) was analyzed by JC-1 staining. siRNA was transfected into the NRK-52E cells to silence NLRP3 expression. RESULTS: CM decreased the viability of NRK-52E cells (P<0.05). CM also elevated the protein levels of cleaved caspase-3, TLR4, NLRP3, IL-1β and IL-18 (P<0.05). Silencing NLRP3 attenuated CM-induced releases of inflammatory cytokines. Moreover, treatment with TLR4 inhibitor TAK-242 or knockdown of NLRP3 by siRNA transfection both attenuated cell apoptosis and loss of MMP caused by CM. CONCLUSION: TLR4/NLRP3 inflammasome takes part in the pathogenesis of CM-induced acute kidney injury, and mediates CM-induced injury and inflammation in renal tubular epithelial cells.     

Effects of magnesium on apoptosis and expression of Foxp3 in peripheral CD4+CD25+ regulatory T cells from asthma patients

AIM:To observe the apoptosis and the expression of forkhead box protein 3(Foxp3) induced by magnesium in CD4⁺CD25⁺ regulatory T cells isolated from healthy and asthmatic human peripheral blood. METHODS:Peripheral blood from healthy volunteers and asthma patients was collected. CD4⁺CD25⁺ T cells were separated by Percoll centrifugation and magnetic separation. The cells were cultured for 72 h and treated with magnesium(10 mmol/L) or control solution. The apoptotic rate and the expression of Foxp3 in the cells were analyzed by flow cytometry. RESULTS:The purity of CD4⁺CD25⁺T cells was 77.4%~92.3% in health group, and was 75.2％~93.8％in asthma group. The proportion of CD4⁺CD25⁺ T cells in CD4⁺T cells was 4.12%~7.98% in healthy adults, and 4.51%~8.68% in asthma patients. No significant difference between the 2 groups was observed. Magnesium at concentration of 10 mmol/L up-regulated the apoptotic rate of CD4⁺CD25⁺ T cells(P<0.05) and did not affect the Foxp3 expression in the cells in both health and asthma groups. CONCLUSION:Magnesium plays therapeutic effects on asthma by inducing the apoptosis of peripheral CD4⁺CD25⁺ regulatory T cells.